Mitogen-activated protein kinases (MAP kinases) mediate multiple cellular pathways regulating growth (1) and differentiation (2, 3). In neuronal cells, MAP kinase activity mediates the actions of growth factors like EGF that stimulate cellular proliferation as well as factors like NGF that maintain neuronal survival and differentiation (4-6). Such ligand-activated signal transduction pathways involve activation of receptor tyrosine kinases which initiates a series of phosphorylation events that activate a cascade of serine/threonine kinases converging on the MAP kinase (also called extracellular signal regulated kinase (ERK)) isoforms, ERK1 and ERK2 (7-9).
Activation of MAP kinase involves specific phosphorylations on threonine and tyrosine residues within the Thr-Glu-Tyr motif (10) by MAP kinase kinase (MAP kinase and ERK kinase or MEK) (2, 11). Phosphorylation of both these residues is required for MAP kinase activation (11, 12). It has been suggested that the inactivation of MAP kinase is a critical event that regulates the physiological response to MAP kinase activation (13). This inactivation is mediated, in part, by dephosphorylation of MAP kinases by dual specificity phosphatases called MKPs (MAP kinase phosphatases) that dephosphorylate both the threonine and tyrosine residues phosphorylated by MEK (13-16). The activation of MAP kinase appears to be tightly regulated through the coordinate action of MEK and MKPs. By regulating the extent of MAP kinase activation, these MKPs may dictate the choice of differentiation or proliferation within a developing cell (17).
The prototype dual-specificity phosphatase, VH1, was identified in vaccinia and showed similarity to cdc25, a protein that controls cell entry into mitosis (18). VH1 homologues from human (PAC-1, CL100, and most recently B23), mouse [MKP-1 (3CH134 or erp)], and yeast (Yop51, MSG5) have also been isolated (19-24.) All are dual-specificity phosphatases that specifically dephosphorylate MAP kinase in vitro (25) and in vivo (13, 15, 26). MKP-1 (also called 3CH134 or erp) was discovered as an immediate early gene whose rapid transcription and subsequent translation are suggested to provide a feed-back loop to terminate growth factor signals (13, 19, 26). Overexpression of mouse MKP-1 was shown to inhibit dramatically fibroblast proliferation suggesting that the inactivation of MAP kinase in vivo by MKP-1 has a profound negative effect on cellular proliferation (25, 26).
MAP kinase activation by growth factors has been extensively studied in PC12 cells (27). PC12 cells originate from a rat pheochromocytoma and retain many features of neural crest-derived cells, most notably the ability to undergo neuronal differentiation upon stimulation by NGF (28). Transfection with activated forms of the oncogenes ras, raf-1 and src into PC12 cells is sufficient for differentiation in the absence of NGF stimulation (6, 8, 29). As each of these genes has been shown to converge on MAP kinase activation, this implies that components of the MAP kinase cascade are required for neuronal differentiation. More recently it has been shown that the activation of MAP kinase kinase, MAPKK-1, is required and sufficient for PC12 cell differentiation (3). Despite our understanding of MAP kinase activation in neuronal differentiation, we know relatively little about MAP kinase inactivation.
Because of the important role of MAP kinases in controlling cell growth and differentiation, it is desirable to have molecular tools useful for inactivation of these enzymes. The present invention presents such tools.